Download Lagos bat virus virulence in mice inoculated by the peripheral route

Epidemiol. Infect. (2009), 137, 1155–1162. f 2009 Cambridge University Press
doi:10.1017/S0950268808001945 Printed in the United Kingdom
Lagos bat virus virulence in mice inoculated by the
peripheral route
W. M A R K O T T E R 1*, I. V. K UZ M IN 2, C. E. R U PP R E C HT 2 A N D L. H. NE L 1
1
Department of Microbiology and Plant Pathology, Faculty of Natural and Agricultural Sciences, University of
Pretoria, South Africa
2
Poxvirus and Rabies Branch, National Center for Zoonotic, Vector-borne and Enteric Diseases, Centers for
Disease Control and Prevention, Atlanta, GA, USA
(Accepted 9 December 2008; first published online 15 January 2009)
SUMMARY
Lagos bat virus (LBV) constitutes genotype (gt) 2 in the Lyssavirus genus. In contrast to the gt1
lyssavirus, rabies virus (RABV), LBV was reported to have markedly reduced levels of peripheral
pathogenicity. However, this opinion was based on a study of one isolate of LBV only and the
reduction in pathogenicity was essentially attributed to the amino-acid substitution at position
333 of glycoprotein ectodomain. In the present study we have demonstrated that peripheral
pathogenicity of representatives of LBV in a murine model is as high as that of RABV.
Comparison of amino-acid substitutions among the viral glycoproteins, demonstrated significant
differences within two antigenic sites between different phylogenetic lineages of LBV. Such
molecular variability potentially contributes to differences in peripheral pathogenicity of
lyssaviruses.
Key words: Africa, glycoprotein, Lagos bat virus, lyssavirus, pathogenicity, rabies.
INTRODUCTION
Lagos bat virus (LBV) constitutes genotype (gt) 2 in
the Lyssavirus genus, family Rhabdoviridae [1]. With
the development of molecular biology techniques, the
Lyssavirus genus was divided into seven genotypes
based on genetic distances. Rabies virus (RABV, gt1)
circulates worldwide, whereas LBV, Mokola virus
(MOKV, gt3) and Duvenhage virus (DUVV, gt4)
have only been isolated from the African continent.
European bat lyssaviruses 1 (EBLV-1, gt5) and 2
(EBLV-2, gt6) are present in Europe, and Australian
* Author for correspondence : Dr W. Markotter, Department of
Microbiology and Plant Pathology, Faculty of Natural and
Agricultural Sciences, University of Pretoria, Pretoria, 0001, South
Africa.
(Email : [email protected])
bat lyssavirus (ABLV, gt7) has been identified from
the Australian continent. All lyssavirus genotypes
have been reported to be pathogenic for animals, and
with the exception of gt2, were also reported to cause
encephalitis in humans. Four new lyssaviruses, currently listed as tentative species in the Lyssavirus genus, were isolated from bats in Eurasia, i.e. Irkut
(IRKV) [2], Aravan (ARAV) [3, 4], Khujand
(KHUV) [4] and West Caucasian bat virus (WCBV)
[2]. Based on phylogeny, serological cross-reactivity
and peripheral pathogenicity to mice, lyssaviruses
were divided into two phylogroups [5]. Phylogroup I
comprised of genotypes 1, 4, 5, 6, 7 (as well as IRKV,
ARAV and KHUV). Phylogroup II includes LBV
and MOKV. Members of phylogroup I have been
shown to be pathogenic for mice when inoculated via
the intracerebral (i.c.) and intramuscular (i.m.) routes.
1156
W. Markotter and others
Table 1. Information about lyssavirus isolates used in experimental infections of mice
Virus name
Genotype
Year of
isolation Geographical origin
Animal species
Reference
WAmyotis
1
2004
Washington, USA
Bat (Myotis sp.)
MOKVSA(252/97)
LBVCAR1974
LBVSA1982
LBVSEN1985
LBVZIM1986
LBVAFR1999
3
2 (Lineage C)
2 (Lineage C)
2 (Lineage A)
2 (Lineage C)
2 (Lineage A)
1997
1974
1982
1985
1986
1999
Cat
Bat (Micropteropus pusillus)
Cat
Bat (Eidolon helvum)
Cat
Bat (Rousettus aegyptiacus)
LagSA2003
LagSA2004
Mongoose2004
LBVSA2006
2 (Lineage C)
2 (Lineage C)
2 (Lineage C)
2 (Lineage C)
2003
2004
2004
2006
South Africa
Central African Republic
South Africa
Senegal
Zimbabwe
Imported into France from an
unknown origin in Africa
South Africa
South Africa
South Africa
South Africa
Present
study
[31]
[32]
[33]
[34]
[35]
[36]
Bat (Epomophorus wahlbergi)
Bat (Epomophorus wahlbergi)
Mongoose (Atilax paludinosus)
Bat (Epomophorus wahlbergi)
[37]
[37]
[38]
[39]
Members of phylogroup II were shown to be pathogenic for mice only when inoculated via the i.c. route
but not when inoculated i.m. Importantly, however,
this result was based on a study of a single isolate of
LBV and a single isolate of MOKV [5]. Members of
phylogroup I cross-neutralize each other. The same is
true for phylogroup II, but very limited crossneutralization was shown between phylogroups I and
II [5]. It has been suggested that WCBV could be
considered as a representative of an independent
phylogroup III in lieu of genetic distance and absence
of serological cross-reactivity with both phylogroups
I and II members [6, 7]. Preliminary pathogenicity
studies indicated that WCBV was pathogenic for mice
when inoculated i.c. but not i.m., as was observed
for phylogroup II, but this virus was pathogenic
for hamsters and bats when inoculated i.m. [7].
Commercial rabies vaccine strains all belong to gt1
and there is no evidence of their lack of efficacy
against any gt1 viruses although they are much less
efficacious against the rabies-related lyssaviruses
(gt2–gt7) [8]. For example, various rabies vaccines
and anti-rabies immune globulins have been shown to
fail to protect animals against MOKV, LBV and
WCBV [6, 9, 10].
The lyssavirus genome codes for five proteins:
Nucleoprotein (N), Phosphoprotein (P), Matrixprotein (M), Glycoprotein (G) and the RNA polymerase (L). G is the most important protein for
interaction of virions with host cell receptors and
for development of humoral immunity [11–16]. In
addition, the Arg/Lys333 amino acid (positively
charged amino acid) in the G protein ectodomain was
identified as being essential for the peripheral
virulence of RABV [14, 16]. Previous genetic analysis
indicated that the Arg/Lys333 is replaced by an Asp333
in phylogroup II lyssaviruses, probably resulting in
their reduced pathogenicity [5]. Amino-acid (aa) substitutions in antigenic site II (aa 34–42 and aa
198–200) of G protein in RABV result in a reduction
of pathogenicity in adult mice when inoculated via the
i.m. route [17].
When complete N, P, M and G genes of 13 LBV
isolates were analysed phylogenetically, the results
identified three different lineages (A–C) of LBV [18].
One of these lineages (lineage A), demonstrated significant sequence diversity, and was suggested as a
new lyssavirus genotype [18]. The present study
was designed to compare the pathogenicity of several
isolates of LBV that represent two of the previously
three identified phylogenetic lineages of this virus
[18], with representatives of RABV and MOKV. The
experiments were performed in a murine model,
comparing different doses of the viruses and routes
of inoculation. Amino-acid substitutions along G
protein, previously suggested to be important for
peripheral pathogenicity of lyssaviruses, were also
compared.
METHODS
Eleven lyssavirus representatives were included in the
present study (Table 1). These isolates were amplified
in suckling mouse brain using i.c. inoculation.
Selection of the LBV representatives was based on
their phylogenetic positions in lineages A and C
(Fig. 1) and their ability to be amplified to significant
titres by mouse inoculation. The single available
Lagos bat virus virulence
RABV (AB009663)
92
gt1
ABLV (NC_003243)
100
1157
gt7
IRKUT (AY333112)
DUVV (AY996322)
91
EBLV-1 (AY863315)
86
gt4
gt5
KHUJAND (AY262023)
56
gt6
EBLV-2 (AY863346)
67
ARAVAN (AY262024)
97
MOKV (S59447)
49
61
62
99
100
100
89
Mongoose2004 (EF547423)
LBVSA1982 (EF547425)
LBVSA2006 (EF547422)
Lineage C
LagSA2003 (EF547421)
LBVZIM1986 (EF547429)
gt2
LBVCAR1974 (EF547430)
LBVNIG1956 (EF547431)
98
gt3
LagSA2004 (EF547423)
LBVAFR1999 (EF547432)
Lineage B
Lineage A
100 LBVSEN1985 (EF547433)
WCBV (AY333113)
0·05
Fig. 1. Neighbour-joining (NJ) phylogenetic tree based upon the 439 amino acids of the ectodomain of the G protein of
representatives of the lyssavirus genus, obtained by the NJ method. GenBank accession numbers are indicated for each
isolate. Bootstrap values are indicated at the nodes and branch lengths are drawn to scale.
isolate of lineage B (LBVNIG1956) was not included
because titres of this virus were low even after extensive passaging in mouse brain and cell cultures. The
presence of lyssavirus antigen in the mouse brains was
confirmed by direct fluorescent antibody test (FAT)
using FITC-labelled monoclonal anti-rabies globulin
(Jujirebio Diagnostics, USA) [19]. Ten percent mouse
brain suspensions were prepared in Minimum
Essential Medium (MEM-10, Gibco, USA) supplemented with 10 % fetal calf serum. The suspensions
were centrifuged at 3200 g for 15 min and stored at
x80 xC. The titre of the inoculum was determined by
i.c. inoculation of virus dilutions into 4-week-old
outbred ICR mice, and the 50% mouse i.c. lethal dose
(MICLD50) was calculated using the Spearman–
Karber method [20].
Four-week-old ICR mice (obtained from Harlan
Sprague–Dawley, USA) were used for experimental
infections. Each mouse was identified with an ear tag
providing a unique number (National Band and Tag
Co., USA). All animal care and experimental procedures were performed in compliance with the Centers for Disease Control and Prevention Institutional
Animal Care and Use Guidelines (USA). The mice
were inoculated with lyssavirus isolates using different routes of inoculation and different doses of
inoculum : group A (102 MICLD50 i.c.), group B
(103 MICLD50 i.m.), group C (10 6 MICLD50 i.m.).
Each group consisted of five mice. Mice were observed for 56 days and clinical signs and mortality
were recorded daily. The i.m. inoculation was performed into the gastrocnemius muscle, in a total volume of 50 ml. The i.c. inoculation was performed in a
total volume of 30 ml as described previously [21]. The
FAT was performed on mouse brain collected from
succumbed or euthanized mice at the end of the experiment on day 56. The nucleotide sequences of the
complete G genes of LBV isolates were generated as
described previously [18]. Nucleotide sequences obtained were assembled and edited using Vector NTI
9.1.0 (Invitrogen, USA) and amino-acid sequences
were deduced using the translate function of this
program. Multiple sequence alignments were generated using the Clustal X program [22].
RESULTS
Susceptibility
The i.c. inoculation of mice with gt1–gt3 lyssavirus
isolates produced similar pathogenicity profiles, all
leading to 100 % mortality (Fig. 2). Intramuscular
1158
W. Markotter and others
100
Deaths (%)
80
60
40
20
gt
LB
3
V
ZI
M
19
LB
86
V
CA
R1
M
97
on
4
go
os
e2
00
4
La
gS
A
20
04
LB
V
SA
19
82
La
gS
A
20
03
LB
V
SA
20
LB
06
V
SE
N
19
LB
85
VA
FR
19
99
gt
1
0
Lyssavirus isolate
Fig. 2. Virulence of genotype (gt) 1 (WAmyotis), gt2 and gt3 [MOKVSA(252/97)] lyssaviruses in 4-week-old mice after
intracerebral (i.c.) and intramuscular (i.m.) inoculation. Results are expressed as a percentage of rabid animals after observation for 56 days. Different viral doses were introduced : (a) , 102 MICLD50 i.c. ; (b) , 103 MICLD50 i.m. ; (c) %, 10 6
MICLD50 i.m.
gt
1
(W
A
Mean incubation time (days)
gt
M
3
YO
(M
TI
O
SS
K
V
PP
SA
)
(2
52
/
LB
97
))
V
ZI
M
LB
19
V
CA 86
R1
M
97
on
4
go
os
e2
00
La
4
gS
A
2
LB
00
4
V
SA
19
82
La
gS
A
20
03
LB
V
SA
20
LB
06
V
SE
LB N1
VA 985
FR
19
99
40
35
30
20
25
15
10
5
0
Lyssavirus isolate
Fig. 3. Mean incubation time (days) of lyssavirus isolates after different routes of inoculation [intracerebral (i.c.) and intramuscular (i.m.)] and different viral doses were introduced into 4-week-old mice. The standard deviation (S.D.) is indicated.
Some S.D. values were 0. (a) %, 102 MICLD50 i.c. ; (b) , 10 6 MICLD50 i.m. ; (c) , 103 MICLD50 i.m.
inoculation with gt1–gt3 lyssaviruses produced more
variable results. When a high dose (106 MICLD50)
was used, all viruses were able to induce disease and
subsequent death, but they were not equally virulent.
At this dose, the RABV (isolate WAmyotis) and
two of the LBV isolates, both from lineage A
(LBVSEN1985 and LBVAFR1999), caused 100 %
fatality rates after i.m. administration of the inoculum. Other LBV isolates injected i.m. at this dose
caused rabies in 20–60% of mice. When a representative of gt3 [isolate MOKVSA(252/97)] was inoculated i.m. at this dose, only 20 % of the mice
succumbed. When viruses were introduced i.m. at a
lower dose (103 MICLD50) the virulence decreased,
and no mice inoculated with isolates MOKVSA(252/
97), LBVCAR1974, LBVSA1982 and LBVSA2006
developed rabies. In contrast, the most significant
virulence was observed for isolates LBVSEN1985 and
LBVSA2004 (60 % and 40 %, respectively).
The duration of incubation periods was also found
to be dependent on the specific virus isolate and the
route of inoculation, and was proportional to the inoculation dose (Fig. 3). The i.c. inoculation produced
the shortest incubation period for all virus isolates
tested. RABV (isolate WAmyotis) did not demonstrate a significant difference in the mean incubation
periods between 102 MICLD50 i.c. and 10 6 MICLD50
i.m., but when introduced i.m. at dose of 103
gt1 (AB009663)
gt4 (AY996322)
gt5 (AY863315)
gt6 (AY863346)
gt7 (NC_003243)
Irkut (AY333112)
Aravan (AY262024)
Khujand (AY262023)
gt3 (S59447)
WCBV (AY333113)
LagSA2003 (EF547421)
LBVSA2006 (EF547422)
Mongoose2004 (EF547423)
LBVSA1982 (EF547425)
LBVZIM1986 (EF547429)
LagSA2004 (EF547428)
LBVCAR1974 (EF547430)
LBVNIG1956 (EF547431)
LBVAFR1999 (EF547432)
LBVSEN1985 (EF547433)
333
330
Lagos bat virus virulence
KSVR
KSVR
KSVR
KSIR
KSVR
KSIR
KSIR
KSVR
KRVD
IKVE
LRVD
LRVD
LRVD
LRVD
LRVD
LRVD
LRVD
LKVD
KRVD
KRVD
Lineage C
1159
Within antigenic site II (positions 34–42 and
198–200), sequence diversity was found at positions
37 and 42 between lineage C isolates and at positions
37, 39, 40, 42, 198 and 200 between the more pathogenic LBV lineage A and the less pathogenic LBV
lineage C. Substitutions were also observed in aa 330,
334 and 336 of antigenic site III between lineage A
and lineage C isolates. No differences were observed
between LBV isolates for other known antigenic
sites within G. When anlaysing other sites on the G
previously implicated as being significant for pathogenicity (positions 164, 182, 205, 210, 242, 255, 268,
303) only position 205 indicated an amino-acid difference between lineage A and lineage C isolates
(Fig. 5).
Lineage B
Lineage A
Fig. 4. Multiple alignment indicating amino acids 330–333
of the ectodomain of the G protein of representatives of the
lyssavirus genus. GenBank accession numbers of the sequences are indicated.
MICLD50, the duration of the incubation period
increased to 17 days. For a representative of gt3
[MOKVSA(252/97)], the mean incubation period increased between the i.c. and i.m. inoculations, and
only the 106 MICLD50 dose caused the disease by the
i.m. route, and in one mouse only. The shortest mean
incubation periods were observed for LBVSEN1985
and LBVAFR1999 for all the routes of inoculation
and over the entire dosage spectrum.
Domains of the lyssavirus G that were previously
implicated in pathogenesis were compared between
viruses used in our study and other representatives
(Figs 4 and 5). Amino-acid positions 330 and 333
on the ectodomain were found to be conserved in
genotypes 1, 4, 5, 6, 7, ARAV, IRKV and KHUV but
not in gt2, gt3 and WCBV. In gt2 and gt3 isolates,
Arg/Lys333 was replaced by an aspartic acid (Asp)
(Fig. 4). With the exception of two lineages (B and C)
of gt2 and WCBV, Lys330 was found to be conserved
within the Lyssavirus genus. The isolates belonging
to lineages B and C of gt2, were found to possess
Leu330. In contrast, isolates belonging to lineage A
of gt2 contain Lys330, similar to all other lyssavirus
genotypes. A multiple alignment of G sequences of
gt2 representatives used in this study is shown in
Figure 5. The lineage A isolates (LBVSEN1985 and
LBVAFR1999) demonstrated significant sequence
diversity in comparison to the other gt2 isolates.
DISCUSSION
The aspect of relative pathogenicity, with reference to
inoculum dose and route of inoculation, is an important criterion in considerations of lyssavirus ecology. Previous studies suggested that phylogroup II
lyssaviruses were not pathogenic when introduced
peripherally. These assertions contributed directly to
the suggestion that such viruses are generally less
pathogenic, and imply that they have limited public
health and veterinary significance [5]. In the present
study we have assessed the susceptibility of mice to
various isolates that are classified within the lyssavirus
gt2 (LBV) in comparison to one isolate each from gt1
(RABV) and gt3 (MOKV). A single isolate of MOKV
and RABV (known to be virulent) were included for
the purpose of comparison. When inoculated i.c., all
lyssaviruses in our panel caused acute progressive
encephalitis (rabies). However, differences were observed when these viruses were inoculated i.m. at
peripheral sites distant to the central nervous system
(CNS).
We have shown that several representatives of LBV
caused rabies in mice when introduced i.m. In the case
of MOKV that was inoculated i.m. at a dose of 106
MICLD50, only 20% of mice succumbed, whereas the
RABV isolate and two LBV isolates were fatal to the
entire respective groups of mice, following the same
dose and route of infection. Of even more significance
was the finding that even at a reduced viral dose (103
MICLD50) inoculated via the i.m. route, six LBV
isolates from lineages A and C demonstrated equal or
greater pathogenicity to mice than did RABV.
Several studies of the lyssavirus G protein suggested specific epitopes that may be involved in
1160
W. Markotter and others
Antigenic site II
Antigenic site Aa 14–19
10
Lineage C
Lineage A
LBVSA1982 (EF547425)
LagSA2003 (EF547421)
LagSA2004 (EF547428)
LBVSA2006 (EF547422)
Mongoose2004 (EF547423)
LBVZIM1986 (EF547429)
LBVCAR1974 (EF547430)
LBVAFR1999 (EF547432)
LBVSEN1985 (EF547433)
DF P LYT I
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
P EK
. . .
. . .
. . .
. . .
. . .
. . .
. .R
. .R
20
I G TWT P
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
. . . . . .
LNP . . .
LNP . . .
30
I D L I H L T C P NN L L S
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
. . . . . .S. . . . . . .
. . . . . .S. . . . . . .
. . . . . .S. . . . . . .
40
GDDGC S D T A T
R. . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
E. . . . . . . . .
E. . . . .N. . .
DA E . . . E . S S
DA E . . . E . S S
50
60
F S Y I E L K T GY
. . . . . . . . . .
. . . . . . . . . .
.G. . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
.N. . . . . . . .
.T.V. . . . .F
.T.V. . . . .F
L T HQKV S G E T
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
.A. . . .P. . .
.A. . . .P. . .
70
C T GVVN E AV T
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . I . . . . .
. . . . I . . . . .
80
Y T N F VGYV T T
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
90
T F KRKH F K P T
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
100
A L A C R D A F HW
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
VS . . . . . . N .
VS . . . . . . N .
Antigenic site II
110
LBVSA1982 (EF547425)
LagSA2003 (EF547421)
LagSA2004 (EF547428)
LBVSA2006 (EF547422)
Mongoose2004 (EF547423)
LBVZIM1986 (EF547429)
LBVCAR1974 (EF547430)
LBVAFR1999 (EF547432)
LBVSEN1985 (EF547433)
K I S GD P RY E E
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
.V. . . . . . . .
.V. . . . . . . .
120
130
140
S L H T P Y P D N S WL R T V T T T K E
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
SLL I
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
I
.
.
.
.
.
.
.
.
SPS
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
IV
. .
. .
. .
. .
. .
. .
. .
. .
150
EMD V Y S R T L H
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . .G. . . .
. . . . .G. . . .
160
L TM F P G G V C S
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . .T. I . .
. . . . .T. I . .
LBVSA1982 (EF547425)
LagSA2003 (EF547421)
LagSA2004 (EF547428)
LBVSA2006 (EF547422)
Mongoose2004 (EF547423)
LBVZIM1986 (EF547429)
LBVCAR1974 (EF547430)
LBVAFR1999 (EF547432)
LBVSEN1985 (EF547433)
MN G S RMC G F T
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
LBVSA1982 (EF547425)
LagSA2003 (EF547421)
LagSA2004 (EF547428)
LBVSA2006 (EF547422)
Mongoose2004 (EF547423)
LBVZIM1986 (EF547429)
LBVCAR1974 (EF547430)
LBVAFR1999 (EF547432)
LBVSEN1985 (EF547433)
L SHF RKLVPG
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
LBVSA1982 (EF547425)
LagSA2003 (EF547421)
LagSA2004 (EF547428)
LBVSA2006 (EF547422)
Mongoose2004 (EF547423)
LBVZIM1986 (EF547429)
LBVCAR1974 (EF547430)
LBVAFR1999 (EF547432)
DQN S L F KKDG
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. KA . V . . . . .
220
DLRGFYP T LK
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
230
240
GACK L T L C CK
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
P G L R L Y D C TW
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
250
V S F T R P E I NV
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . .H.
. . . . . . . .H.
Antigenic site III
310
320
Y L KAY T I I NG
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . .L. .
. . . . . . .L. .
410
420
D A D D F V E V HM
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . .D. . .
330
S LME T N V H Y L
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . .K
. . . . . . . . .K
T N H D Y T LWL P
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
190
200
E D A N K S MA C D
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. .P. . .LI . .
. .P. . .LI . .
I
.
.
.
.
.
.
.
.
F V T S T GKK S
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
. I . . . . . . .
. . . . . .R.A
. . . . . .R.A
260
WC S P N Q L V N V
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . .D. . . . .
. . . .D. . . . .
270
HNN P L D E I E H
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . I . . . . .
. . . . I . . . . .
280
L I VGD L I RK P
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . .D. . . . . .
. . .D. . . . . .
290
EECLDTLET I
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . .V
. . . . . . . . .V
300
LM S K S I S F R R
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . .L. . . .
. . . . .L. . . .
Antigenic site V
340
RVD S L ND I L P
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . .N.V. . . .
. . .N.V. . . .
KFYP S S P SCP
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . I . . .A
. . . . . I . . .A
180
Antigenic site IV
Antigenic site I
210
170
350
S K G C L KMN K Q
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . V . NK
. . . . . . V . NK
360
CVD S YKGV F F
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . .R. . . .
. ME . D T . . . .
. ME . D T . . . .
NG I
. . .
. . .
. . .
. . .
. . .
. . .
. . .
. . .
370
I KG L DGH
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . .H. . .
. . .P. .R
. . .P. .R
LI
. .
. .
. .
. .
. .
. .
. .
. .
380
I
.
.
.
.
.
.
.
.
P EMQ S S L
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . .G.
. . . . .G.
390
L K Q LMD L L K A
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
400
AV F P L RH P L I
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
430
P D I QK L I S DV
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . .V. . .
D L G L P SWS L
. . . . . . . . .
. . . . . . . . .
. . . . . . . . .
.F. . . . . . .
. . . . . . . . .
. . . . . . .G.
. . . . .N. .F
Fig. 5. Multiple alignment indicating differences in the G protein of genotype 2 representatives analysed in pathogenicity
studies. Antigenic sites, as well as domains previously indicated as playing a role in pathogenesis are indicated.
pathogenicity. For example, mutations in the G protein can affect pathogenesis as indicated by the aminoacid replacement at position 333 of the ectodomain.
The presence of a positively charged amino acid in
this position, Arg or Lys, led to a virulent phenotype
of RABV while mutations to a Gln, Ile, Gly, Met or
Ser led to a less pathogenic or avirulent virus [11, 12,
14, 16]. The aa 333 mutation could also affect the rate
of viral spread from cell to cell [23]. A double mutation of aa 330 and aa 333 led to a further reduction
of pathogenicity of RABV compared to a single aa
333 substitution [12, 15]. A recombinant virus with a
Glu333 reverted back to a more pathogenic phenotype
when Asn194 mutated to Lys194 during suckling mouse
brain passage [24]. Badrane et al. [5] reported limited
peripheral pathogenicity of phylogroup II lyssaviruses, presumably related to the Asp333 in their glycoprotein ectodomain. From the results of our study
it is evident that Asp333 is not the sole determinant of
reduced pathogenicity since LBV isolates from lineage
A (Asp333) demonstrated the same peripheral pathogenicity to mice as a RABV (Arg333) isolate. The
significance of Lys330 within lineage A representatives (similar to the phylogroup I lyssaviruses) distinguishes them from the other LBV lineages, and is
difficult to assess. For example, Lys330 is also present
in the MOKV isolate that demonstrated reduced
peripheral pathogenicity in our study. However,
we only analysed one isolate of MOKV, and future
studies including different MOKV representatives may
provide a better resolution. Mutations in antigenic
site II of the G has previously been shown to render a
less pathogenic laboratory RABV strain CVS when
introduced i.m. into adult mice [17]. When comparing
the amino-acid sequence of antigenic site II of LBV
isolates analysed in this study, differences were observed between isolates of lineages A and C. A difference between isolates of lineages A and C was
also observed in aa 255, which has previously been
implicated as being essential in the pathogenicity of
Lagos bat virus virulence
the virulent Nishigahara strain (gt1) [16]. Our results
suggest that certain LBV representatives, particularly
from phylogenetic lineage A (which was recently
suggested for consideration as an independent genotype [18]), demonstrate the same or even greater
peripheral pathogenicity to mice, as a RABV representative and suggest that the pathogenicity of
phylogroup II lyssaviruses has been underestimated.
Variation in pathogenicity can occur within a genotype, depending on the strain origin and animal model
[25–29]. Even if a mouse is a standard laboratory
species for lyssavirus infection, this is not the natural
lyssavirus host, and therefore probably not the best
model for detailed pathogenesis studies. Indeed, all
available phylogroup II isolates were obtained from
naturally infected wild and domestic mammals, indicating that these viruses have well established
pathways for natural circulation. Intradermal exposures could in some cases be worthy of consideration since LBV is associated with large fruit bats,
e.g. Rousettus, Epomophorus and Eidolon [18] from
which a bite can easily access mammalian muscles.
Our findings clearly indicate the need for improved
surveillance and public health precautions for phylogroup II lyssaviruses. Considering that commercially
available rabies vaccines do not protect against
phylogroup II lyssaviruses, new biologicals which
would be capable of protecting against such viruses
[6, 8], are needed and have been shown to be feasible,
at least experimentally [30].
ACKNOWLEDGEMENTS
The authors thank Dr C. T. Sabeta (Agricultural
Research Council, Onderstepoort Veterinary Institute, Rabies Unit, South Africa) for providing the
MOKVSA(252/97) and LBVSA1982 isolate and
Dr F. Cliquet [Agence Française de Sécurité Sanitaire
des Aliments (AFSSA), France] for providing the
LBV1999AFR isolate. This study was supported in
part by the National Research Foundation of South
Africa, the University of Pretoria International
Affairs Officee’s Postgraduate Study Abroad Bursary
Programme, and the US National Vaccine Program
Office. Use of trade names and commercial sources
are for identification only and do not imply endorsement by the U.S. Department of Health and Human
Services. The findings and conclusions in this report
are those of the authors and do not necessarily represent the views of the funding agencies.
1161
D E C L A R A T I O N OF IN T E R E S T
None.
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